Tension-sensitive Plk1 phosphorylation on BubR1 regulates the stability of kinetochore–microtubule interactions

Abstract

Mitotic phosphorylation of the spindle checkpoint component BubR1 is highly conserved throughout evolution. Here, we demonstrate that BubR1 is phosphorylated on the Cdk1 site T620, which triggers the recruitment of Plk1 and phosphorylation of BubR1 by Plk1 both in vitro and in vivo. Phosphorylation does not appear to be required for spindle checkpoint function but instead is important for the stability of kinetochore-microtubule (KT-MT) interactions, timely mitotic progression, and chromosome alignment onto the metaphase plate. By quantitative mass spectrometry, we identify S676 as a Plk1-specific phosphorylation site on BubR1. Furthermore, using a phospho-specific antibody, we show that this site is phosphorylated during prometaphase, but dephosphorylated at metaphase upon establishment of tension between sister chromatids. These findings describe the first in vivo verified phosphorylation site for human BubR1, identify Plk1 as the kinase responsible for causing the characteristic mitotic BubR1 upshift, and attribute a KT-specific function to the hyperphosphorylated form of BubR1 in the stabilization of KT-MT interactions.[Keywords: BubR1; tension; phosphorylation; spindle assembly checkpoint] Supplemental material is available at http://www.genesdev.org. The BubR1 protein was first identified as an essential and evolutionarily conserved component of the spindle assembly checkpoint (SAC). During mitosis, this checkpoint inhibits the anaphase-promoting complex/cyclosome (APC/C), a large E3 ubiquitin ligase, so that chromosome segregation is triggered only after bipolar attachment to the spindle microtubules (MTs) has been achieved (Musacchio and Hardwick 2002;Bharadwaj and Yu 2004;Taylor et al. 2004). SAC dysfunction and the consequential premature APC/C activation can result in aneuploidy, a hallmark of human solid tumors (Kops et al. 2005). Throughout prophase and prometaphase, SAC components including BubR1 are enriched at kinetochores (KTs), complex proteinaceous structures assembled on centromeric DNA, where a "wait anaphase" signal is thought to be generated. KTs represent the major point of contact between mitotic spindle MTs and chromosomes (Maiato et al. 2004;Chan et al. 2005). Several proteins and multiprotein complexes, including BubR1 ( In addition, KT-associated proteins contribute to maintain SAC activity until the last chromosome has undergone bipolar attachment. Although the exact nature of SAC signaling is not well understood, biochemical and genetic evidence indicate that the source of this signal is at the centromere/KT, where tension generated upon amphitelic attachment is sensed (Pinsky and Biggins 2005;Baumann et al. 2007). How such a mechanical property is transduced into a biochemical signaling cascade remains unclear, but there is strong evidence that phosphorylation plays a key role (Gorbsky 1995).Along with APC/C-mediated proteolyis, M-phase progression is controlled through phosphorylation by mitotic kinases. In addition to the key regulator cyclindependent kinase 1 (Cdk1), Polo-like ...

Centrobin regulates the assembly of functional mitotic spindles

“…Control and Centrobin-depleted cells were probed for BubR1, a marker of spindle checkpoint activation, and BubR1 S676 phosphorylation, a site that is phosphorylated in prometaphase and remains so until tension is established during metaphase (Elowe et al, 2007). These markers stained kinetochores strongly in both control and Centrobin-depleted prometaphase cells.…”

“…The BubR1 Ser676 antibody was a kind gift from S Elowe and was used as described previously (Elowe et al, 2007). The NuMA antibody was a kind gift from D Compton and was used as described previously (Gaglio et al, 1995).…”

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

“…All constructs were verified by sequencing. The target sequences of different siRNAs are siBubR1 (GGAGATCCTCTACAAAGGG, nucleotides 1126-1144), siMps1-1 (CTTGAATCCCTGTGGAAAT, nucleotides 2627-2646), and siMps1-2 (CGGAATTCATTGAGACAAA, nucleotides 766-784), which have been described previously and synthesized by Qiagen (48,66). All the plasmids and siRNAs were transfected into cells using Lipofectamine 2000 (Invitrogen) according to the user's manual.…”

A role for Drosophila Polo protein in chromosome resolution and segregation during mitosis

“…7 In fact, because Rod streaming is dependent on bipolar attachment of the chromosomes 7 our results are suggestive that the kinetochore-microtubule attachment is defective after Polodownregulation, in agreement with increasing evidence that Polo-like kinases are required for stabilization of kinetochore-microtubule attachments (reviewed in ref. 10). It should be noticed that tension also contributes to the stabilization of the kinetochore-microtubule attachments and it was shown that 3F3/2 staining at kinetochores, a sensor of bipolar tension, is affected in polo mutants.…”

“…9 Furthermore, mammalian Plk1 was shown to be responsible for the tension-sensitive phosphorylation on BubR1. 10 Polo is required for chromosome resolution. The chromosome missegregation phenotype of Polo-depleted cells resemble the ones described for lack of SMC4 (structural maintenance chromosome 4) and Topoisomerase II, proteins required for chromosome resolution.…”